RESEARCH ARTICLE 1231
Development 140, 1231-1239 (2013) doi:10.1242/dev.084665 © 2013. Published by The Company of Biologists Ltd
Myod and H19-Igf2 locus interactions are required for diaphragm formation in the mouse Maud Borensztein1, Paul Monnier1, Franck Court2, Yann Louault1, Marie-Anne Ripoche1, Laurent Tiret3, Zizhen Yao4, Stephen J. Tapscott4, Thierry Forné2, Didier Montarras5 and Luisa Dandolo1,* SUMMARY The myogenic regulatory factor Myod and insulin-like growth factor 2 (Igf2) have been shown to interact in vitro during myogenic differentiation. In order to understand how they interact in vivo, we produced double-mutant mice lacking both the Myod and Igf2 genes. Surprisingly, these mice display neonatal lethality due to severe diaphragm atrophy. Alteration of diaphragm muscle development occurs as early as 15.5 days post-coitum in the double-mutant embryos and leads to a defect in the terminal differentiation of muscle progenitor cells. A negative-feedback loop was detected between Myod and Igf2 in embryonic muscles. Igf2 belongs to the imprinted H19-Igf2 locus. Molecular analyses show binding of Myod on a mesodermal enhancer (CS9) of the H19 gene. Chromatin conformation capture experiments reveal direct interaction of CS9 with the H19 promoter, leading to increased H19 expression in the presence of Myod. In turn, the non-coding H19 RNA represses Igf2 expression in trans. In addition, Igf2 also negatively regulates Myod expression, possibly by reducing the expression of the Srf transcription factor, a known Myod activator. In conclusion, Igf2 and Myod are tightly co-regulated in skeletal muscles and act in parallel pathways in the diaphragm, where they affect the progression of myogenic differentiation. Igf2 is therefore an essential player in the formation of a functional diaphragm in the absence of Myod.
INTRODUCTION In mammals, several myogenic regulatory factors (MRFs), such as Myf5, Mrf4 (Myf6), Myod and myogenin (Myog), are involved in skeletal muscle development. Myod1 (Myod), which was the first gene of this family to be identified, plays an essential role in the determination and differentiation of the skeletal muscle lineage (Pownall et al., 2002; Rudnicki et al., 1993). Mice carrying a targeted deletion of Myod are viable and fertile but they display a growth reduction phenotype of 20% and deficient muscular regeneration compared with their wild-type (wt) littermates (Megeney et al., 1996; Rudnicki et al., 1992). Muscles of the diaphragm, as well as limb and tongue, originate from a pool of myogenic migrant precursor cells derived from the hypaxial dermomyotome (Sambasivan and Tajbakhsh, 2007). Under the control of Pax3, these precursors delaminate from the ventrolateral lips of the dermomyotome, migrate and reach the primary diaphragm at around embryonic day (E) 12.5. These cells then express Myod and Myf5; they actively proliferate and fully colonize the diaphragm, with the exception of the tendinous central region. Embryonic and fetal waves of myogenesis induce the production of muscle fibers under the control of Myog to produce a functional diaphragm (Buckingham, 2007). 1
Genetics and Development Department, Inserm U1016, CNRS UMR 8104, University of Paris Descartes, Institut Cochin, 75014 Paris, France. 2Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, University of Montpellier II, 34293 Montpellier, France. 3UMR 955 de Génétique Fonctionnelle et Médicale, Institut National de la Recherche Agronomique, University of Paris-Est, Ecole Nationale Vétérinaire d’Alfort, 94700 Maisons-Alfort, France. 4Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. 5 Molecular Genetics of Development Unit, Department of Developmental Biology, URA CNRS 2578, Institut Pasteur, 75015 Paris, France. *Author for correspondence (
[email protected]) Accepted 1 January 2013
Interestingly, analysis of mutants of the different MRFs has provided some insight into their importance in the development of the diaphragm. Myod mutants display a delay in hypaxial development that is compensated by the presence of Myf5, as shown by the lethality of Myod;Myf5 double-knockout mutants (Kablar et al., 1998; Kablar et al., 1997). When Myod mutants are bred on an mdx (Dmd) background this also results in death of the newborn pups. They show no alteration of limb muscles but display severe atrophy of the diaphragm (Kablar et al., 2003). In this case, Myf5dependent myogenic precursors are able to compensate for the absence of Myod in the hypaxial limb muscles but not completely in the diaphragm. Myod and Myf5 are both responsible for the establishment and maintenance of the lineage, although they each have specific roles, particularly in the diaphragm. Myog mutants also display neonatal lethality and show a strong reduction in the size of the diaphragm (Hasty et al., 1993; Nabeshima et al., 1993). In this mutant, the precursors are present but their differentiation is severely compromised. Growth and skeletal muscle development are also controlled by other factors, such as members of the insulin-like growth factor (Igf) family. Igf2 is one of the major growth factors implicated in embryonic growth, cell survival and the differentiation of several tissues (Smith et al., 2006). Mice lacking Igf2 are viable and fertile but display a growth reduction of 40% compared with their wt littermates (DeChiara et al., 1990). In vitro studies have shown that Igf2 protein plays a role in myoblast proliferation and differentiation (Rotwein, 2003). Interestingly, a link between the Myod and Igf2 genes was shown in myoblast cell culture (C2 cell line) (Montarras et al., 1996). Further studies suggested that Igf2, through binding to the Igf1 receptor (Igf1r), activates the Akt pathway and Myod downstream targets, although the exact mechanism has not been elucidated (Wilson and Rotwein, 2006; Woelfle et al., 2005). Recently, a microRNA, miR-483-5p, the gene for which is embedded in intron 2 of Igf2, has been shown to target the 3⬘UTR
DEVELOPMENT
KEY WORDS: Diaphragm differentiation, Genomic imprinting, Myogenesis
Development 140 (6)
1232 RESEARCH ARTICLE
of serum response factor (Srf) (Qiao et al., 2011). The transcription factor Srf is one of the factors responsible for the activation of Myod expression (Gauthier-Rouviere et al., 1996; L’honore et al., 2003). Nevertheless, little is known in vivo about the role of Igf2 in skeletal muscle development and about potential interactions between Myod and Igf2. Both genes are expressed during embryogenesis, but after birth Igf2 is strongly downregulated, to be replaced postnatally by Igf1 (Rotwein, 2003). Igf2 belongs to the imprinted H19-Igf2 locus and displays monoallelic expression from the paternally inherited allele. H19 is expressed from the maternal allele and produces a 2.3 kb noncoding RNA, the function of which has not been fully determined (Gabory et al., 2010; Leighton et al., 1995), as well as a microRNA, miR-675 (Smits et al., 2008). Interestingly, H19 was identified in the same selective screen as Myod and called at the time MyoH (Davis et al., 1987). The main control element of this locus is the differentially methylated imprinting control region (ICR) located between Igf2 and H19. Both genes are coordinately expressed in many embryonic tissues of mesoderm and endoderm origin. This expression is under the control of two sets of enhancers located downstream of H19 that act as endoderm- or mesoderm-specific enhancers. In addition to cis effects mediated by the enhancers and the ICR, it was recently shown that the H19 non-coding RNA could act in trans by downregulating the expression of Igf2 (Gabory et al., 2009; Wilkin et al., 2000). In order to investigate the link between Igf2 and Myod, we developed a mouse model combining defective alleles for both genes. The double-mutant (DM) mice lacking Myod and Igf2 surprisingly displayed neonatal lethality, whereas the Myod and Igf2 single knockouts were viable. We observed severe atrophy and absence of contraction of the diaphragm, whereas all other muscles displayed no obvious defects. We investigated the mechanisms linked to the non-functionality of the diaphragm in the DM embryos by studying its structural anomalies. Analysis of E13.5-18.5 diaphragms revealed a striking lack of terminal differentiation of the diaphragm of DM embryos. We then studied the molecular pathways linked to Igf2 and Myod and identified a negativefeedback loop between these two genes. H19 expression was also affected by the binding of Myod on the mesodermal enhancers that control this locus. Finally, Srf expression was affected by the lack of Igf2, suggesting a role for this growth factor in the control of Myod expression. These findings demonstrate a tight interaction between Igf2 and Myod in myogenesis, with the existence of a compensation mechanism by which overexpression of Igf2 can compensate for the absence of Myod and vice versa. As shown previously in other contexts, the diaphragm differs strikingly from other skeletal muscles and our results reveal an essential role for Igf2, in the absence of Myod, in the production of a functional diaphragm.
H19Δ3 mice harbor a 3 kb deletion of the H19 gene and were bred on the 129/Sv background (Ripoche et al., 1997). H19Δ3 females were bred with Myod−/− males and the resulting heterozygous females were backcrossed to Myod−/− males. The resulting progeny were collected at E18.5, genotyped to select H19–/+;Myod+/+ (n=6) and H19–/+;Myod–/+(n=6) embryos and limb muscles and diaphragm were collected.
MATERIALS AND METHODS
Srf ChIP experiments
Mouse strains
ChIP assays were performed using the HighCell ChIP Kit (Diagenode) with modifications to the manufacturer’s instructions to adapt for ChIP on tissues. Limb muscles of wt postnatal day (P) 0 pups were dissected, minced and fixed in 1% formaldehyde. Tissues were disrupted using a Dounce tissue grinder. Chromatin was sonicated into 200-800 bp fragments using a Bioruptor (Diagenode). Input DNA was purified from 1% of the chromatin. Total chromatin was used for immunoprecipitation, using protein G-coated magnetic beads (Diagenode) and 2 µg Srf antibody (Santa Cruz, SC335 X) or 2 µg non-specific rabbit IgG (negative control). DNA was purified and analyzed by quantitative PCR (qPCR). Il4 intron was used as a negative control. Primers are listed in supplementary material Table S1.
DNA was extracted from tail biopsies and PCR was performed with GoTaq polymerase (Promega) according to manufacturer’s instructions. Primers are described in supplementary material Table S1. Embryo collection, muscle histology and immunohistochemistry
E12.5-18.5 embryos were collected from the uterus of Myod+/− and Myod−/− females and weighed. E18.5 embryos were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Sections (5 µm) were deparaffinized in xylene, rehydrated and stained with Hematoxylin and Eosin (H&E). Diaphragms from E13.5, E15.5 and E18.5 embryos were removed and embedded in gelatin-sucrose, frozen in isopentane cooled in liquid nitrogen, and sectioned using a microtome cryostat (Leica). For assessment of tissue morphology, 5 µm transverse sections were stained with H&E. Fiber size was analyzed by immunostaining muscle sections with an antibody to laminin (Novocastra) and counterstaining with DAPI. Fiber cross-sectional area was determined using MetaMorph software (Molecular Devices). We analyzed myoblasts and muscle differentiation by immunostaining with Pax7 (Santa Cruz) and myogenin (Dako) antibodies on 7 µm transverse sections counterstained with DAPI. The number of positive cells was determined using MetaMorph and ImageJ (NIH) software. Diaphragm contraction study
After caesarian delivery at E18.5, newborns were collected and dissected to access the phrenic nerve. Microelectrodes were used to stimulate the phrenic nerve in order to activate the diaphragm with a frequency of 20 Hz, a pulse of 10 milliseconds and 0.5-1.5 V. For wt and Myod+/−, n=8; Igf2+/−, n=1; Myod−/−, n=2; DM, n=6. Electron microscopy
Electron microscopy was performed on dissected diaphragms from E18.5 wt, single-mutant and DM embryos as previously described (Schmitt et al., 2001). Whole-mount skeletal staining
E18.5 fetuses were quickly boiled, skinned and eviscerated. They were fixed in 95% ethanol for 3 days, and then placed for 24 hours in Alcian Blue solution [15 mg Alcian Blue 8GX (Sigma) in 80 ml 95% ethanol and 20 ml glacial acetic acid] at 4°C for cartilage staining. Embryos were rinsed for 2 days in 95% ethanol, then cleared in 1% KOH for 2 hours at 4°C, and counterstained with Alizarin Red solution (5 mg Alizarin Red (Sigma) in 100 ml 1% KOH) for 3 hours at 4°C for bone staining. Embryo clearing was completed in the following ratios of 1% KOH to glycerol: 80:20, 60:40, 40:60, 20:80. ChIP-Seq experiments showing Myod binding
Myod ChIP-Seq was performed on myoblasts and myotubes in S. Tapscott’s laboratory. Results were extracted from published raw data (Cao et al., 2010).
DEVELOPMENT
All experimental designs and procedures were in agreement with guidelines of the animal ethics committee of the Ministère de l’Agriculture (France). H19Δ13 mice were bred on a C57BL/6 background. Myod−/− mice were bred on a C57BL/6/CBA outbred background and maintained mainly as heterozygotes. The Igf2−/− strain was on a 129/Sv background. Matings were between Myod+/− females and Myod+/−;Igf2+/− males, and embryos were collected at E12.5-18.5 (day of plug was considered E0.5). Matings between a Myod+/− female and a heterozygous Myod+/−;Igf2+/− male produce the four genotypes of interest in the same litter: wild type (wt), Igf2+/−, Myod−/− and the Myod−/−;Igf2+/− double mutant (DM).
Genotyping
Myod and H19-Igf2 in diaphragm formation Nuclei extraction from myoblasts
−/−
Limb muscles of E18.5 wt and Myod embryos were dissected and dissociated in a solution of 0.75 u/ml collagenase (Roche), 1.2 u/ml dispase (Roche), 2.5 mM CaCl2. Collected cells were filtered twice on 70 μm and 45 μm filters and washed in PBS. Cells were resuspended and lysed in 0.8% NP40 buffer with 0.3 M sucrose, then loaded on a 1.2 M sucrose solution and centrifuged for 20 minutes at 8500 rpm (8000 g). Pelleted nuclei were resuspended in 40% glycerol buffer. Chromatin conformation capture (3C)
3C-qPCR experiments were performed on wt and Myod−/− nuclei extracted from myoblasts as described previously (Hagège et al., 2007). Interaction frequencies were determined at BamHI sites surrounding the H19 locus. 3C products were quantified on a LightCycler 480 II apparatus (Roche) and data were normalized according to a published algorithm (Braem et al., 2008). Primers are listed in supplementary material Table S2. Gene expression analysis
Collected tissues were disrupted using a MixerMill apparatus (Qiagen) and total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Extracted RNA was RQ1 DNase treated (Promega) and then re-extracted with phenol:chloroform and chloroform before ethanol precipitation. For the expression profile analysis, reverse transcription with SuperScript II reverse transcriptase (Invitrogen) was carried out on 500 ng total RNA with random hexamer oligonucleotides. Quantitative real-time PCR (RT-qPCR) was performed for Myod, Srf, skeletal actin (Acta1) and cardiac actin (Actc1), Igf2 and H19 on 10 ng cDNA in 10 μl final volume with FastStart MasterMix reagent (Roche) in a LightCycler 2.0 apparatus (Roche). The level of gene expression was normalized to the geometric mean of the expression level of Tbp and Gapdh housekeeping genes with geNorm software (v3.4) (Vandesompele et al., 2002). RNA was prepared from five diaphragms and five limb muscles of E18.5 embryos per genotype. Three independent reverse transcription experiments were carried out for each sample. Detection of miR-483-5p was performed using a stem-loop primer for the reverse transcription step and two primers to detect the level of expression of the microRNA in different samples. Data were normalized to the U6 RNA, using TaqMan microRNA assays according to the manufacturers guidelines (Applied Biosystems). Primers are listed in supplementary material Table S1.
RESEARCH ARTICLE 1233
Igf2+/− mutants, suggesting that removing one copy of Myod had no synergistic effect on the deleterious Igf2 phenotype. Myod−/− embryos displayed a weight phenotype identical to wt embryos. The decrease in weight of 20% that was previously described for the Myod mutants was detected only after birth (supplementary material Fig. S1). Therefore, it appeared that Igf2 affected the growth of the embryo as early as E12.5, whereas only postnatal growth was affected by the Myod defect. In consequence, the DM embryos display the same weight phenotype as Igf2+/− embryos. Caesarian delivery and genotyping at the end of gestation showed that out of 60 newborns (from eight Myod−/− or Myod+/− mothers), all live pups that survived were either wt, heterozygous or singlemutant pups (n=48), whereas all pups that precociously died were DMs (n=12). DM pups were myotonic, became rapidly cyanotic and exhibited respiratory failure, leading to their death. To evaluate whether they breathed, lungs were collected and immediately placed on a water disk. They sank to the bottom of the tube, which indicated that alveoli had never inflated, in contrast to their healthy littermates (Fig. 1A). Functional and structural anomalies of the diaphragm of DM embryos Experiments were performed to evaluate in vivo diaphragm contraction in E18.5 embryos of the different genotypes. The
Statistical analysis
RESULTS Neonatal lethality and atrophy of the Igf2;Myod double-mutant diaphragm In order to study the possible interactions between Igf2 and Myod in vivo, we produced a mouse model combining defective alleles for both genes. Because Igf2 is imprinted, a paternal heterozygous mouse (Igf2mat+/pat–) produces no Igf2 protein. We performed matings between heterozygous or homozygous Myod females (Myod+/−;Igf2+/+ or Myod−/−;Igf2+/+) and heterozygous Myod+/−;Igf2+/− males to obtain the four genotypes of interest: wt, Igf2+/−, Myod−/− and the Myod−/−;Igf2+/−double mutant (DM). Surprisingly, no viable DMs were found in the litters, suggesting embryonic or neonatal lethality of Myod−/−;Igf2+/− individuals. Embryos collected at different stages showed complete viability of the DMs until the end of fetal development (E18.5) (supplementary material Fig. S1). Embryos of each genotype were weighed between E12.5 and E18.5. In Igf2+/− mutants, the deficit in weight previously detected at E18.5 (−40%), in fact occurs as early as E12.5 (−20%) (DeChiara et al., 1990). Myod+/−;Igf2+/− embryos were identical to
Fig. 1. Diaphragm characteristics in E18.5 mutants. (A) Lungs were dissected out from the mouse embryos after caesarian delivery and dropped into water. Igf2+/− lungs float, whereas those from the Myod−/−;Igf2+/− DM sink to the bottom, which means that DM lungs were not inflated with air. (B) Sagittal sections of E18.5 diaphragms stained with H&E. (C) Immunostaining for laminin on E18.5 diaphragm sections was used to assess the distribution (%) of fiber cross-sectional area in the four genotypes. au, arbitrary units. The inset illustrates the curve graph of the histogram. (D) E18.5 diaphragm sections immunostained for laminin were used to assess muscle fiber number in the four genotypes. *P
